Phytopathology®

‘ Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening disease, is transmitted by the Asian citrus psyllid, Diaphorina citri, in a propagative, circulative, and persistent manner. Unfortunately, ‘ Ca. L. asiaticus’ is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (wt/vol) sucrose and 0.1% ascorbic acid (wt/vol) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (10 nymphs per seedling) for an inoculation period of 2 weeks. A transmission rate of up to 80% was recorded at 6 months postinoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of ‘ Ca. L. asiaticus’ becomes available.

The unculturable bacterium ‘ Candidatus Liberibacter solanacearum’ (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid ( Bactericera cockerelli). However, the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype and other potentially novel CLso variants are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by ‘ Candidatus Liberibacter asiaticus’ ( CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited pathogen associated with devastating diseases in members of the Solanaceae and Apiaceae and vectored by several psyllid species. Different Lso haplotypes have been identified, and LsoA and LsoB are responsible for diseases in Solanaceae crops. Our efforts are aimed at identifying pathogenicity factors used by this bacterium to thrive in different hosts. Bacterial secreted proteins can play a role in host colonization or the manipulation of the host immune responses; these proteins are called effectors. In this study, we identified six LsoB-specific proteins with a conserved secretion motif as well as a conserved N-terminal domain in the mature protein. These proteins had different expression and secretion patterns but a similar subcellular localization in Nicotiana benthamiana leaves, suggesting that they play different roles regardless of their conserved secretion motif. One of these proteins, CKC_04425, was expressed at high levels in the insect vector and the host plant, indicating that it could play a role in both the plant and insect hosts, whereas the others were mainly expressed in the plant. One protein, CKC_05701, was able to efficiently suppress programmed cell death and reactive oxygen species production, suggesting that it may have a virulence role in LsoB-specific pathogenesis.

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and is fatal to young trees and mature trees of certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: citrus growers need treatments that improve tree health, fruit production, and most importantly, economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to the discovery of those solutions and to estimate the likelihood of success of a treatment aimed at commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions; understanding the strengths and limitations of each assay, as well as how best to compare different assays, is critical for decision-making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a reevaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops.

‘ Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant−pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant−pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.

‘ Candidatus Liberibacter solanacearum’ is an insect-transmitted bacterium associated with several plant diseases. In the Mediterranean Basin, ‘ Ca. L. solanacearum’ haplotype D is vectored by Bactericera trigonica and can severely infect carrot plants leading to abnormal growth phenotypes and significant yield losses. Insecticide applications are insufficient to suppress disease spread and damage, and additional means for disease control are needed. In the current study, we evaluated the resistance of 97 carrot accessions to the bacterial pathogen ‘ Ca. L. solanacearum’ and its associated symptoms. Accessions (Western and Asian types) were first screened in two commercial carrot fields. We found that Western type accessions were less prone to develop disease symptoms in both fields and were less frequently visited by the insect vector in one field. Overall, 22 Asian and five Western accessions with significantly lower disease incidence compared with the commercial cultivar were found. These accessions were then inoculated with ‘ Ca. L. solanacearum’ under controlled conditions and were assessed for disease incidence, insect oviposition, and bacterial relative titer. Five accessions (three Asian and two Western) had significantly lower disease incidence compared with the reference cultivar. Interestingly, disease incidence was not necessarily in line with insect oviposition or in planta bacterial titer, which may indicate that other, perhaps physiological, differences among the accessions may govern the susceptibility of plants to the disease. The resistant accessions found in this study could be used in future resistance breeding programs and to better understand the underlying mechanisms of resistance to ‘ Ca. L. solanacearum’.

The Asian citrus psyllid, Diaphorina citri, is an invasive insect and a vector of ‘ Candidatus Liberibacter asiaticus’ ( CLas), a bacterium whose growth in Citrus species results in huanglongbing (HLB), also known as citrus greening disease. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification and nevertheless contain significant quantities of host DNA. To overcome these hurdles, we developed a simple pretreatment DNase and filtration (PDF) protocol to remove host DNA and directly sequence CLas and the complete, primarily uncultivable microbiome from D. citri adults. The PDF protocol yielded CLas abundances upward of 60% and facilitated direct measurement of CLas and endosymbiont replication rates in psyllids. The PDF protocol confirmed our lab strains derived from a progenitor Florida CLas strain and accumulated 156 genetic variants, underscoring the utility of this method for bacterial strain tracking. CLas genetic polymorphisms arising in lab-reared psyllid populations included prophage-encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest that laboratory propagation of CLas could result in different phenotypic trajectories among laboratories and could confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can directly inform agricultural management strategies related to bacterial strain tracking, insect microbiome surveillance, and antibiotic resistance screening.

‘Candidatus Phytoplasma pruni’ infection in cherries causes small, misshapen fruit with poor color and taste, rendering the fruit unmarketable. However, this is a disease with a long development cycle and a scattered, nonuniform symptom distribution in the early stages. To better understand the biology as well as the relationship between pathogen titer and disease expression, we carried out seasonal, spatial, and temporal examinations of ‘Ca. P. pruni’ titer and distribution in infected orchard-grown trees. Sequential sampling of heavily infected trees revealed marked seasonal patterns, with differential accumulation in woody stem and leaf tissues and, most notably, within fruit in the early stages of development from bloom to pit hardening. Furthermore, mapping phytoplasma distribution and titer in trees at different stages of infection indicated that infection proceeds through a series of stages. Initially, infection spreads basipetally and accumulates in the roots before populating aerial parts of the trees from the trunk upward, with infection of specific tissues and limbs followed by an increasing phytoplasma titer. Finally, we observed a correlation between phytoplasma titer and symptom severity, with severe symptom onset associated with three to four orders of magnitude more phytoplasma than mild symptoms. Cumulatively, these data aid in accurate sampling and management decision-making and furthers our understanding of disease development.